CN110691796B - Antibody FC variants for increasing blood half-life - Google Patents

Abstract

The present invention relates to polypeptides comprising an Fc variant or antibodies comprising the same, wherein a portion of the amino acid sequence of the Fc domain of a human antibody is replaced with a different amino acid sequence. The Fc variants of the invention can be used in a variety of antibodies and Fc fusion constructs. In one aspect, the antibodies or Fc fusion constructs of the invention are agents, preferably therapeutic agents, for use in therapy, diagnosis or research. The Fc variants of the invention can maximize in vivo half-life by optimizing a portion of the amino acid sequence and are useful in the treatment of cancer. The antibodies and Fc fusion constructs of the invention are useful for killing target antigens, such as target cells containing cancer cells. Alternatively, the antibodies and Fc fusion constructs of the invention are used to block, antagonize, or interfere with a target antigen, such as to antagonize a cytokine or cytokine receptor, for example.

Description

Antibody FC variants for increasing blood half-life

Technical Field

The present invention relates to novel antibody Fc variants having increased blood half-life and methods for preparing the antibody Fc variants.

Background

With recent advances in biotechnology such as gene recombination and cell culture, extensive studies have been made on the structure and function of proteins worldwide. Biotechnology has prompted people to better understand important phenomena and plays a decisive role in elucidating pathogenesis of various diseases to pave the way for effective diagnosis and treatment of diseases, thereby greatly improving quality of life. In particular, since the hybridoma technology for producing monoclonal antibodies by fusing B cells with myeloma cells was developed in 1975 (Kohler and Milstein, nature,256:495-497, 1975), immunotherapy using therapeutic antibodies has been widely studied and developed in clinical applications including cancer, autoimmune diseases, inflammation, cardiovascular diseases, and infection.

Compared with the existing small molecule drugs, the therapeutic antibody has higher specificity to the target, lower biotoxicity and fewer side effects. Another advantage of therapeutic antibodies is their long blood half-life (about 3 weeks). Because of these advantages, therapeutic antibodies are considered to be the most effective treatment of cancer. Indeed, large pharmaceutical companies and research institutions have focused their research and development capabilities on therapeutic antibodies that specifically bind and effectively remove cancer cells including oncogenes. Rogowski, ann's, qingsheng, yabang and BMS are the major pharmaceutical companies currently developing therapeutic antibody drugs. In particular, rogowski developed three novel therapeutic antibodies for anticancer therapy, herceptin, avastin and rituximab, which were sold in the 2012 at the cost of 195 billion dollars, and obtained considerable profits on the global market, currently leading the global market for antibody drugs. As the sales of the species gram (Remicade) increases, the predatory company developing the species gram is rapidly expanding in the global antibody market. It is well known that other pharmaceutical companies such as yaban and BMS possess many therapeutic antibodies in the final stages of development. As a result, biomedical science including therapeutic antibodies specific to the target disease with few side effects is rapidly replacing small molecule drugs that dominate the global pharmaceutical market.

The Fc region of an antibody recruits immune leukocytes or serum complement molecules and then triggers the clearance of defective cells such as tumor cells or infected cells. The Fc interface between the C.gamma.2 and C.gamma.3 domains mediates interactions with neonatal Fc receptors (FcRn) and their binding allows endocytic antibodies to circulate from the endosome back into the blood stream (Raghavan et al, 1996,Annu Rev Cell Dev Biol 12:181-220; ghetie et al, 2000,Annu Rev Immunol 18:739-766). This process, coupled with the inhibition of renal filtration due to the large size of the full length IgG antibody molecule, extends the half-life of the antibody serum from 1 week to 3 weeks. Furthermore, fc binding to FcRn plays a key role in antibody transport. Thus, the Fc region is critical for extending serum persistence of circulating antibodies through intracellular transport and circulation mechanisms.

Administration of an antibody or Fc-fusion protein as a therapeutic agent requires a predetermined injection frequency in view of the half-life of the therapeutic agent. Longer in vivo half-life may reduce the frequency of injection or reduce the dosage. Thus, in many clinical studies currently being conducted, much effort has been focused on developing next-generation anti-cancer therapeutic antibodies and anti-cancer therapeutic proteins by introducing mutations into the Fc domain to increase the half-life of the antibody or introducing variants into the Fc domain to achieve maximum ADCC effects (Modified from Cancer Immunol res.2015/Thomson Reuters).

However, although the research group is working to develop some proteins and antibodies with enhanced binding affinity for FcRn, and to extend the in vivo half-life by introducing some mutations into the Fc domain, a significant increase in vivo half-life has not been achieved. Under these circumstances, development of an optimally mutated antibody is urgently required.

The description of the background is provided only for the sake of a better understanding of the background of the invention and should not be construed as corresponding to the prior art known to those skilled in the art.

Detailed Description

Problems to be solved by the invention

The present inventors have earnestly studied to effectively increase the in vivo half-life of existing therapeutic proteins or antibodies, and as a result, have found that the therapeutic proteins or antibodies can be optimized by replacing a part of the amino acid sequence of the wild-type Fc domain with a different amino acid sequence, so that the blood half-life can be maximally prolonged while maintaining their excellent activity.

It is an object of the present invention to provide a polypeptide comprising an Fc variant produced by replacing a portion of the amino acid sequence of a human antibody Fc domain with a different amino acid sequence.

It is another object of the present invention to provide an antibody comprising said polypeptide.

It is another object of the present invention to provide a nucleic acid molecule encoding said polypeptide.

It is another object of the present invention to provide a vector comprising said nucleic acid molecule.

It is a further object of the present invention to provide a host cell comprising said vector.

It is another object of the invention to provide a composition comprising a polypeptide, an antibody, a nucleic acid molecule or a vector.

It is another object of the invention to provide a method for producing a polypeptide or an antibody.

It is another object of the invention to provide a method of screening polypeptides.

Other objects and advantages of the present invention will become more apparent from the detailed description, claims and drawings herein.

Means for solving the problems

In one aspect, the invention provides a polypeptide comprising an Fc variant produced by replacing a portion of the amino acid sequence of a human antibody Fc domain with a different amino acid sequence.

Another aspect of the invention provides a composition for increasing the blood half-life of a therapeutic antibody or protein, the composition comprising an Fc variant produced by replacing a portion of the amino acid sequence of a human antibody Fc domain with a different amino acid sequence.

The present inventors have sought to find a method that effectively increases the in vivo half-life of existing therapeutic proteins or antibodies, and as a result have found that a therapeutic protein or antibody comprising an Fc variant that is produced by substituting and optimizing a portion of the amino acid sequence of a wild-type Fc domain with a different amino acid sequence can achieve the maximum in vivo half-life.

Antibodies are proteins that specifically bind to a particular antigen. Natural antibodies are heterodimeric glycoproteins having a molecular weight of about 150000 daltons, typically consisting of two identical light chains (L) and two identical heavy chains (H).

The human antibodies used in the present invention fall into one of five main categories: igA, igD, igE, igG and IgM. The human antibody is preferably an IgG antibody. Antibodies were digested with papain to produce two Fab fragments and one Fc fragment, while the Fc region of human IgG molecules was produced by papain digestion of the N-terminal Cys226 (Deisenhofer, biochemistry 20:2361-2370, 1981).

The antibody Fc domain may be IgA, igM, igE, igD or an IgG antibody or modified Fc domain thereof. In one embodiment, the domain is an Fc domain of an IgG antibody, e.g., an Fc domain of an IgG1, igG2a, igG2b, igG3, or IgG4 antibody. In one embodiment, the Fc domain may be an IgG1 Fc domain, e.g., an Fc domain of an anti-HER 2 antibody, preferably an Fc domain of trastuzumab, more preferably having the amino acid sequence of SEQ ID NO:28, and an Fc domain of the sequence shown. The polypeptides of the invention may optionally be partially or fully glycosylated. In addition to an Fc domain, a polypeptide of the invention may also include one or more regions derived from an antibody. In addition, the polypeptides of the invention may include antigen binding domains derived from antibodies, and may form antibodies or antibody-like proteins with another polypeptide.

Here, the amino acid residues of the Fc domain of antibodies are named according to the Kabat EU numbering system commonly used in the art, as described in Kabat et al, "Sequences of Proteins of Immunological Interest" 5 th edition, U.S. Pat. No. HEALTH AND Human Services, NIH Publication No.91-3242, 1991.

According to a preferred embodiment of the invention, the substituted Fc variant comprises M428L as amino acid substitution according to the Kabat EU numbering system.

According to a preferred embodiment of the invention, the substituted Fc variant comprises a) M428L and b) Q311R or L309G as amino acid substitutions according to the Kabat EU numbering system.

According to a preferred embodiment of the invention, the substituted Fc variant comprises P228L and M428L as amino acid substitutions according to the Kabat EU numbering system.

According to a preferred embodiment of the invention, the Fc variant comprising the amino acid substitutions P228L and M428L comprises further amino acid substitutions at one or more positions selected from the group consisting of position 234, 264, 269, 292, 309, 342, 359, 364, 368, 388, 394, 422, 434 and 445 according to the Kabat EU numbering system.

Additional amino acid substitutions may be L309R and N434S.

Additional amino acid substitutions may be V264M, L368Q, E388D, V D and P445S.

Additional amino acid substitutions may be R292L, T359A and S364G.

Additional amino acid substitutions may be L234F, E269D, Q342L, E388D and T394A.

According to a preferred embodiment of the invention, the substituted Fc variant comprises a) M428L and b) P230Q or P230S as amino acid substitutions according to the Kabat EU numbering system.

According to a preferred embodiment of the invention, the substituted Fc variant comprising amino acid substitutions a) M428L and b) P230Q or P230S according to the Kabat EU numbering system comprises additional amino acid substitutions at one or more positions selected from positions 243, 246, 295, 320, 356, 361, 384 and 405.

Additional amino acid substitutions may be F243Y, K246,246, 246E, N361,361S and N384I.

Additional amino acid substitutions may be Q295L, K320M, D356E and F405I.

The present invention relates to Fc variants comprising one or more amino acid substitutions that modulate the binding and dissociation of Fc variants to neonatal Fc receptors (FcRn). In particular, the Fc variants of the invention or functional variants thereof show an increased binding affinity for FcRn under acidic conditions (pH below 7) and very low binding to FcRn under neutral pH conditions.

The therapeutic antibody whose half-life is to be increased is not particularly limited, and examples thereof include polyclonal antibodies, monoclonal antibodies, minibodies, domain antibodies, bispecific antibodies, antibody mimics, chimeric antibodies, antibody conjugates, human antibodies, humanized antibodies, and fragments thereof.

As monoclonal antibodies, for example, human antibodies such as panitumumab (victimb), ofatuzumab (Arzerra), golimumab (euphorib), and ipilimab (Yervoy) and the like; humanized antibodies, such as tolizumab (yamero), trastuzumab (herceptin), bevacizumab (avastin), omalizumab (sorel), meperimumab (Bosatria), gemtuzumab (mailita), palizumab (Synagis), ranibizumab (noradapted), cetuzumab (Cimzia), oreuzumab, mogamulizumab (Poteligeo), and eculizumab (Shu Lirui); and chimeric antibodies, such as rituximab (rituximab), cetuximab (erbitux), infliximab (rickettsia), and basiliximab (sultam).

There is no particular limitation on the therapeutic protein whose half-life is to be increased, examples of which include: hormones, such as insulin; cytokines such as growth factors, interferons, interleukins, erythropoietin, neutrophil growth factors and transforming growth factors; fc fusion proteins such as etanercept (enli), abamectin (Ai Liya, zaltrap), abamectin (oregano), alfasin (Amevive), beraceep (Nulojix), and cilexetil (Arcalyst); therapeutic peptides such as teriparatide (futaolone), exenatide (berda), liraglutide (norand li), lanreotide (cable Ma Dulin), pramlintide (Symlin) and enfuwei peptide (Fuzeon); and polypeptides including part or all of the VEGF receptor, her2 receptor, G protein coupled receptor, and ion channel cell surface receptor.

The half-life of a therapeutic antibody or protein may be extended by binding to a polypeptide of the invention or a nucleic acid encoding the polypeptide or by introducing into a vector expressing the nucleic acid.

According to a preferred embodiment of the invention, the Fc variant has an increase in binding affinity for FcRn of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% or at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold or at least 100-fold over the binding affinity of the wild-type Fc domain for FcRn at a pH of 5.6 to 6.4 (preferably 5.8 to 6.2).

According to a preferred embodiment of the invention, the extent of dissociation of the Fc variant from the neonatal Fc receptor (FcRn) may be the same or not substantially altered as the extent of dissociation of the wild-type Fc domain from the neonatal Fc receptor (FcRn) at a pH of 7.0 to 7.8 (preferably 7.2 to 7.6).

According to one embodiment of the invention, the displaced Fc variant exhibits a higher binding affinity under weakly acidic conditions (e.g., at a pH of 5.8 to 6.2) than the wild-type Fc or other developed Fc variants, and which dissociates to the same or substantially the same or higher extent under neutral conditions (e.g., at a pH of 7.4) as the wild-type Fc or other developed Fc variants (see example 4 and example 8).

According to a preferred embodiment of the invention, the substituted Fc variant has a long half-life compared to the wild type.

The half-life of a substituted Fc variant according to the invention may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% longer than the wild-type Fc domain, or at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold or at least 10-fold longer than the wild-type Fc domain.

According to one embodiment of the invention, the substituted Fc variants have significantly improved in vivo half-life compared to the wild type (see example 11 and table 3).

As used herein, "fcγreceptor" or "fcγr" refers to any member of the family of proteins that bind to the Fc region of an IgG antibody and are encoded by fcγr genes. Examples of such fcγreceptors or fcγrs include, but are not limited to: fcyri (CD 64), including fcyria, fcyrib, and fcyric; fcγrii (CD 32), including fcγriia, fcγriib, and fcγriic; fcγriii (CD 16), including fcγriiia and fcγriiib; and undiscovered fcγr. Fcγr can be derived from mammalian organisms including humans, mice, rats, rabbits and monkeys, among other organisms.

As used herein, "FcRn" or "neonatal Fc receptor" refers to a protein that binds to the Fc region of an IgG antibody and is at least partially encoded by the FcRn gene. FcRn can be derived from mammalian organisms including humans, mice, rats, rabbits, and monkeys, among other organisms. Functional FcRn proteins include two polypeptides called the heavy and light chains. The light chain is a beta-2-microglobulin and the heavy chain is encoded by the FcRn gene.

In another aspect of the invention, an antibody comprising a polypeptide is provided.

As used herein, the term "antibody" refers to polyclonal antibodies, monoclonal antibodies, microantibodies, domain antibodies, bispecific antibodies, antibody mimics, chimeric antibodies, antibody conjugates, human antibodies, humanized antibodies, or fragments thereof (e.g., antigen-binding antibody fragments).

According to a preferred embodiment of the invention, the half-life of the Fc domain or the polypeptide comprising the Fc domain may be maximized by optimizing the corresponding antibody Fc region (e.g.M 428L and Q311R; or M428L and L309G).

In another aspect the invention provides a nucleic acid molecule encoding the polypeptide, a vector comprising the nucleic acid molecule or a host cell comprising the vector.

The nucleic acid molecules of the invention may be isolated or recombinant nucleic acid molecules. Examples of such nucleic acids include single and double stranded DNA and RNA and their corresponding complementary sequences. The isolated nucleic acid may be isolated from a natural source. In this case, the isolated nucleic acid is separated from the peripheral gene sequences present in the genome of the subject from which the nucleic acid is to be isolated. An isolated nucleic acid is understood to be a nucleic acid, such as a PCR product, a cDNA molecule or an oligonucleotide, which is synthesized enzymatically or chemically from a template. In this case, the nucleic acid resulting from the procedure can be understood as an isolated nucleic acid molecule. An isolated nucleic acid molecule represents a nucleic acid molecule in the form of an isolated fragment or as part of a larger nucleic acid construct. A nucleic acid is "operably linked" when it is arranged in a functional relationship with another nucleic acid sequence. For example, when expressed as a preprotein, the DNA of the preprotein is operably linked to the DNA of the polypeptide, which preprotein is the polypeptide prior to secretion. Promoters or enhancers that affect transcription of a polypeptide sequence are operably linked to a coding sequence, or ribosome binding sites are operably linked to a coding sequence, which when so arranged, facilitate translation. Generally, the term "operably linked" refers to DNA sequences that are to be linked being adjacent to each other. For a secretory leader, the term "operably linked" refers to the secretory leader being present adjacent to each other in the same leader frame. The required enhancers are not necessarily contiguous. Ligation is performed by ligation at convenient restriction enzyme sites. In the absence of such sites, synthetic oligonucleotide adaptors or linkers are used in accordance with suitable methods known in the art.

As used herein, the term "vector" is used to refer to a vector into which a nucleic acid sequence may be inserted for introduction into a cell so that replication may take place. The nucleic acid sequence may be "exogenous" or "heterologous". Vectors include plasmids, cosmids, and viruses (e.g., phage). The vector can be constructed by standard recombinant techniques by those skilled in the art, which are described in Maniatis et al, molecular Cloning, A Laboratory Manual, cold Spring Harbor Press, cold Spring Harbor, N.Y.,1988; and Ausubel et al, in: current Protocols in Molecular Biology, john, wiley & Sons, inc, NY, 1994).

As used herein, the term "expression vector" refers to a vector containing a nucleic acid sequence encoding at least a portion of a gene product capable of being transcribed. In some cases, the RNA molecule is subsequently translated into a protein, polypeptide, or peptide. Expression vectors may contain various "control sequences". In addition to control sequences that control transcription and translation, vectors and expression vectors may contain nucleic acid sequences that have other functions.

As used herein, the term "host cell" refers to any transgenic organism capable of replicating a vector or expressing a gene encoded by a vector. Suitable organisms include eukaryotes and prokaryotes. The host cell may be transfected or transformed with the vector. Transfection or transformation refers to a process for transferring or introducing an exogenous nucleic acid molecule into a host cell.

The host cell of the present invention is preferably a bacterial cell, a CHO cell, a HeLa cell, a HEK293 cell, a BHK-21 cell, a COS7 cell, a COP5 cell, an A549 cell or a NIH3T3 cell, but is not limited thereto.

In another aspect of the invention, there is provided a method for producing a polypeptide comprising a variant Fc of a human antibody, comprising: a) Culturing a host cell comprising a vector comprising a nucleic acid molecule encoding the polypeptide; and b) harvesting the polypeptide expressed by the host cell.

In another aspect of the invention, there is provided a method for preparing an antibody comprising: a) Culturing a host cell expressing an antibody comprising the polypeptide; b) Purifying antibodies expressed by the host cells.

In the method of the present invention, the antibody may be purified by filtration, HPLC, anion or cation exchange, high Performance Liquid Chromatography (HPLC), affinity chromatography, or a combination thereof, preferably affinity chromatography using protein a.

In another aspect of the invention, there is provided a method for screening a polypeptide comprising an Fc variant, comprising: constructing a library comprising M428L as a mutated Fc variant according to the Kabat EU numbering system; and b) selecting from the Fc variants comprising the M428L mutation an Fc variant having a higher affinity for FcRn than the wild type at a pH of 5.6 to 6.4.

An Fc variant comprising an M428L mutation may comprise at least one additional amino acid substitution.

According to a preferred embodiment of the invention, the further amino acid substitution comprises Q311R or L309G as mutation.

According to a preferred embodiment of the invention, the further amino acid substitution comprises P228L as mutation.

Fc variants comprising the P228L mutation may comprise at least one additional amino acid substitution.

The additional amino acid substitution is not particularly limited, but is preferably an amino acid mutation at least one position selected from the group consisting of positions 234, 264, 269, 292, 309, 342, 359, 364, 368, 388, 394, 422, 434 and 445 according to the EU numbering system of Kabat.

According to a preferred embodiment of the invention, the further amino acid substitutions comprise P230Q or P230S as mutations.

Fc variants comprising P230 mutations may comprise at least one additional amino acid substitution.

The additional amino acid substitution is not particularly limited, but is preferably an amino acid mutation at least one position selected from the group consisting of positions 243, 246, 295, 320, 356, 361, 384 and 405 according to the Kabat EU numbering system.

The screening methods of the invention may use Fluorescence Activated Cell Sorting (FACS) or automated flow cytometry. Flow cytometry instruments are well known to those skilled in the art. Examples of such instruments include FACSaria, FACS Star Plus, FACScan and FACSort (Becton Dickinson, foster City, calif.), epics C (Coulter Epics division, hairyearly, florida), MOFLO (Cytomation of Colorado Prain, colorado) and MOFLO-XDP (Beckman Coulter, indianapolis, indiana). Flow cytometry generally involves the separation of cells or other particles in a liquid sample. In general, the purpose of flow cytometry is to analyze isolated particles for one or more characteristics, such as the presence of a labeled ligand or other molecule. The particles pass the sensor one after the other and are sorted according to size, refractive index, light scattering, opacity, roughness, shape, fluorescence, etc.

In another aspect of the invention, there is provided a composition comprising a polypeptide, antibody, nucleic acid molecule or vector, said polypeptide comprising an Fc variant having one or more than one amino acid substitution.

According to a preferred embodiment of the present invention, the composition is a pharmaceutical composition for preventing or treating cancer.

According to a preferred embodiment of the invention, the pharmaceutical composition (or polypeptide, antibody, nucleic acid molecule or vector) recognizes a cancer antigen.

According to one embodiment of the invention, the Fc variants have Antibody Dependent Cellular Cytotoxicity (ADCC) activity comparable to or higher than that of the control group (e.g., trastuzumab), thereby achieving significantly increased half-life and high anticancer activity (see example 13 and fig. 18).

The pharmaceutical compositions of the invention may comprise (a) a polypeptide, an antibody, a nucleic acid molecule encoding the polypeptide or a vector comprising the nucleic acid molecule and (b) a pharmaceutically acceptable carrier.

In yet another aspect, the invention provides a method for preventing or treating cancer comprising administering a pharmaceutical composition to a subject.

The type of cancer prevented or treated by the method of the present invention is not limited. The pharmaceutical compositions of the invention can be administered to treat a number of cancers, including leukemias and lymphomas such as acute lymphoblastic leukemia, acute non-lymphoblastic leukemia, chronic myelogenous leukemia, hodgkin's disease, non-hodgkin's lymphoma, multiple myeloma, childhood solid tumors such as brain tumor, neuroblastoma, retinoblastoma, nephroblastoma, bone tumor and soft tissue sarcoma, common solid tumors in adults such as lung cancer, breast cancer, prostate cancer, urinary cancer, uterine cancer, oral cancer, pancreatic cancer, melanoma and other skin cancers, stomach cancer, ovarian cancer, brain tumor, liver cancer, laryngeal cancer, thyroid cancer, esophageal cancer and testicular cancer.

The pharmaceutically acceptable carrier of the pharmaceutical composition according to the present invention may be any carrier known in the art. Examples of carriers suitable for use in the pharmaceutical compositions of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starches, acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. The pharmaceutical composition of the present invention may further comprise at least one additive selected from the group consisting of lubricants, wetting agents, sweeteners, flavoring agents, emulsifying agents, suspending agents and preserving agents. Details of suitable pharmaceutically acceptable carriers and formulations can be found in Remington's Pharmaceutical Sciences (19 th edition, 1995).

The pharmaceutical compositions of the present invention may be administered orally or parenterally, preferably parenterally. For example, the pharmaceutical compositions of the present invention may be administered by intravenous, topical or intraperitoneal injection.

The subject is not particularly limited but is preferably construed to include vertebrates, more preferably primates including humans and chimpanzees, domestic pets including dogs and cats, domestic animals including cows, horses, sheep and goats, and rodents including mice and rats.

The appropriate dosage of the pharmaceutical composition according to the present invention depends on various factors such as the dosage form, the mode of administration, the age, weight, sex and pathological condition of the patient, diet, the time and route of administration, the rate of excretion and responsiveness. The effective dosages of the pharmaceutical compositions according to the present invention for the desired treatment or prophylaxis can be readily determined and prescribed by one of ordinary skill in the art. According to a preferred embodiment of the present invention, the daily dose of the pharmaceutical composition according to the present invention is from 0.0001mg/kg to 100mg/kg.

The pharmaceutical compositions of the invention may be prepared in unit dosage form or dispensed in multi-dose containers with pharmaceutically acceptable carriers and/or excipients by suitable methods readily performed by those of ordinary skill in the art. The pharmaceutical compositions of the present invention may be in the form of solutions, suspensions or emulsions in oily or aqueous vehicles. The pharmaceutical composition of the present invention may be in the form of an extract, powder, granule, tablet or capsule. The pharmaceutical composition of the present invention may further comprise a dispersing agent or a stabilizing agent.

The pharmaceutical composition of the present invention may be used in monotherapy. Or the pharmaceutical composition of the present invention may be used in combination with usual chemotherapy or radiotherapy. This combination therapy is more effective for cancer treatment. Chemotherapeutic agents that may be used with the compositions of the present invention include cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, busulfan, nitrosourea, actinomycin D, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide, tamoxifen, paclitaxel, anti-platinum, 5-fluorouracil, vincristine, vinblastine, methotrexate, and the like. Radiation therapies that may be used with the compositions of the present invention include X-ray radiation and gamma-ray radiation.

Effects of the invention

The features and advantages of the present invention are summarized below.

(I) The present invention provides a polypeptide comprising an Fc variant produced by replacing a portion of the amino acid sequence of a human antibody Fc domain with a different amino acid sequence.

(Ii) The invention also provides methods of producing a polypeptide or an antibody comprising the polypeptide.

(Iii) The Fc variants of the invention are useful in the treatment of cancer because their in vivo half-life can be maximized by optimizing a portion of the amino acid sequence.

Drawings

FIG. 1 shows expression vectors for expression and purification of tetrameric FcRn and dimeric FcRn, and SDS-PAGE gels after purification.

FIG. 2 is a schematic of a library constructed so that 18 amino acids are included at positions M252 and M428.

FIG. 3 shows the 2M library retrieval process and the sorted M428L variant.

FIG. 4 schematically shows error and spot libraries constructed based on M428L.

FIG. 5 shows FACS fluorescence intensities of variants sorted from (5 a) error library and (5 b) spot library.

Figure 6 shows plasmids for expressing trastuzumab heavy and light chains in animal cells.

Fig. 7 shows the expression and purification results of wild-type trastuzumab.

FIG. 8 compares the physical properties of commercial trastuzumab and internal trastuzumab (a: CE-cIEF, b: SEC).

FIG. 9 compares the physical properties of commercial trastuzumab and internal trastuzumab by N-glycan profiling.

FIG. 10 shows the expression and purification results of 10 trastuzumab Fc variants (a: affinity chromatography, b: SDS-PAGE analysis, c: final yield list).

Fig. 11 shows SEC characterization results of trastuzumab Fc variants.

Figure 12 shows the binding of trastuzumab Fc variants to FcRn, as measured by ELISA.

FIG. 13 shows the binding of trastuzumab Fc variants to hFcRn at pH values of 6.0 and 7.4, as measured using a BiaCore instrument (a: pH6.0 (capture method) b: pH7.4 (avid format)).

Figure 14 compares the pharmacokinetics of commercial trastuzumab and internal trastuzumab in conventional mice (C57 BL/6J (B6)) and human FcRn Tg mice.

Fig. 15 shows the results of pharmacokinetic analysis of Fc variants in human FcRn Tg mice (after intravenous injection of variants (5 mg/kg each), n=5).

Fig. 16 shows binding force of trastuzumab Fc variants to fcγr, as measured by ELISA.

Figure 17 compares the effector function of trastuzumab Fc variants with that of normal IgG, trastuzumab as a control (ADCC assay).

Figure 18 compares the effector functions (ADCC) of trastuzumab Fc variants.

Fig. 19 shows the binding of trastuzumab Fc variants to C1q, as measured by ELISA.

Best mode for carrying out the invention

The invention will be elucidated in more detail with reference to the following examples. It will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention.

Examples

Example 1: expression and purification of neonatal Fc receptor (FcRn) for searching Fc variant libraries

Tetrameric FcRn and dimeric FcRn for retrieving Fc variants with improved pH-dependent binding to FcRn were expressed and purified. For this purpose, an expression vector was prepared (FIG. 1). pMAZ-beta 2 microglobulin-GS linker-FcRnα chain-streptavidin-His was constructed as a DNA plasmid to obtain tetrameric FcRn. The DNA was co-transfected into HEK 293F cells and transiently expressed at a level of 300 ml. The resulting culture medium was centrifuged at 7000rpm for 10 minutes. The collected supernatants were equilibrated with 25 XPBS and filtered with a 0.2 μm bottle top filter (Merck Millipore). After equilibration with PBS, fcRn was allowed to bind to Ni-NTA resin (Qiagen) for 16h at 4 ℃. FcRn binding resin was loaded onto the column and eluted with 50ml of wash buffer No. 1 (PBS), 25ml of wash buffer No. 2 (pbs+10 mM imidazole), 25ml of wash buffer No. 3 (pbs+20 mM imidazole) and 200 μl of wash buffer No. 4 (pbs+250 mM imidazole) to remove proteins other than tFcRn. Then, 2.5ml of elution buffer (PBS+250 mM imidazole) was passed through the column to obtain tFcRn. The buffer was replaced with new buffer using a centrifugal filter device (Merck Millipore). Dimeric FcRn was obtained from pcDNA-fcrnα chain-GST- β2 microglobulin plasmids obtained from oslo university. The DNA was co-transfected into HEK 293F cells and transiently expressed at a level of 300 ml. The resulting culture medium was centrifuged at 7000rpm for 10 minutes. The collected supernatants were equilibrated with 25 XPBS and filtered with a 0.2 μm bottle top filter (Merck Millipore). After equilibration with PBS, fcRn was allowed to bind glutathione sepharose 4B (incospharm) for 16h at 4 ℃. FcRn binding resin was loaded onto the column and the column eluted with 10ml wash buffer (PBS) to remove proteins other than dFcRn. Then, 2.5ml of elution buffer (50 mM Tris-HCl+10mM GSH, pH 8.0) was passed through the column. The buffer is replaced with a new buffer using the centrifugal filter device 3K (Merck Millipore). The sizes of the purified tetrameric and dimeric FcRn were determined using SDS-PAGE gels (fig. 1). Purified tetrameric FcRn and dimeric FcRn were fluorescently labeled with Alexa 488 for fluorescence detection.

Example 2: construction of Fc variant 2M libraries

PMopac12-NlpA-Fc-FLAG was constructed from the gene of the Fc domain of trastuzumab (SEQ ID NO: 29) using SfiI restriction endonuclease. Library inserts were constructed based on vectors using pMopac12-seq-Fw、Fc-M252-1-Rv、Fc-M252-2-Rv、Fc-M252-3-Rv、Fc-M428-Fw、Fc-M428-1-Rv、Fc-M428-2-Rv、Fc-M428-3-Rv、Fc-M428-frg3-Fw、 and pMopac12-seq-Rv primers such that two Met residues in the Fc were replaced with 18 different amino acids except Cys and Met (table 1 and figure 2). The inserts were treated with SfiI restriction enzyme and ligated to the same SfiI treated vector. The ligated inserts were then transformed into E.coli Jude1((F'[Tn10(Tet^r)proAB⁺lacI^qΔ(lacZ)M15]mcrAΔ(mrr-hsdRMS-mcrBC)Φ80dla cZΔM15 ΔlacX74 deoR recA1 araD139Δ(ara leu)7697 galU galKrpsLendA1nupG) to construct a large 2M library of Fc variants (library size: 1X 10 ⁹).

TABLE 1

pMopac12-seq-Fw	5`-CCAGGCTTTACACTTTATGC-3`
		Fc-M252-1-Rv	5`-CCTCAGGGGTCCGGGAGATGWAGAGGGTGTCCTTGGGTTTTGGG-3`
Fc-M252-2-Rv	5`-CCTCAGGGGTCCGGGAGATKNBGAGGGTGTCCTTGGGTTTTGGG-3`
		Fc-M252-3-Rv	5`-CCTCAGGGGTCCGGGAGATCCAGAGGGTGTCCTTGGGTTTTGGG-3`
Fc-M428-Fw	5`-ATCTCCCGGACCCCTGAGG-3`
		Fc-M428-1-Rv	5`-GTAGTGGTTGTGCAGAGCCTCATGGWACACGGAGCATGAGAAGACGTTCC-3`
Fc-M428-2-Rv	5`-GTAGTGGTTGTGCAGAGCCTCATGKNBCACGGAGCATGAGAAGACGTTCC-3`
		Fc-M428-3-Rv	5`-GTAGTGGTTGTGCAGAGCCTCATGCCACACGGAGCATGAGAAGACGTTCC-3`
Fc-M428-frg3-Fw	5`-CATGAGGCTCTGCACAACCACTAC-3`
		pMopac12-seq-Rv	5`-CTGCCCATGTTGACGATTG-3`
Fc-Sub#0-Rv	5`-GTCCTTGGGTTTTGGGGGGAAG-3`
		Fc-Sub#1-1-Fw	5`-CTTCCCCCCAAAACCCAAGGACNNKCTCATGATCTCCCGGACCCCTGAGGTCACATGCG-3`
Fc-Sub#1-2-Fw	5`-CTTCCCCCCAAAACCCAAGGACACCNNKATGATCTCCCGGACCCCTGAGGTCACATGCG-3`
		Fc-Sub#1-3-Fw	5`-CTTCCCCCCAAAACCCAAGGACACCCTCATGNNKTCCCGGACCCCTGAGGTCACATGCG-3`
Fc-Sub#1-4-Fw	5`-CTTCCCCCCAAAACCCAAGGACACCCTCATGATCNNKCGGACCCCTGAGGTCACATGCG-3`
		Fc-Sub#1-5-Fw	5`-CTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCNNKACCCCTGAGGTCACATGCG-3`
Fc-Sub#1-Rv	5`-GACGGTGAGGACGCTGACC-3`
		Fc-Sub#2-1-Fw	5`-GGTCAGCGTCCTCACCGTCNNKCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGG-3`
Fc-Sub#2-2-Fw	5`-GGTCAGCGTCCTCACCGTCCTGCACNNKGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGG-3`
		Fc-Sub#2-3-Fw	5`-GGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGNNKAATGGCAAGGAGTACAAGTGCAAGG-3`
Fc-Sub#2-Rv	5`-CACGGAGCATGAGAAGACGTTCC-3`
		Fc-Sub#3-1-Fw	5`-GGAACGTCTTCTCATGCTCCGTGCTGCATNNKGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTG-3`
Fc-Sub#3-2-Fw	5`-GGAACGTCTTCTCATGCTCCGTGCTGCATGAGGCTNNKCACAACCACTACACGCAGAAGAGCCTCTCCCTG-3`
		Fc-Sub#3-3-Fw	5`-GGAACGTCTTCTCATGCTCCGTGCTGCATGAGGCTCTGCACNNKCACTACACGCAGAAGAGCCTCTCCCTG-3`
Fc-Sub#3-4-Fw	5`-GGAACGTCTTCTCATGCTCCGTGCTGCATGAGGCTCTGCACAACCACNNKACGCAGAAGAGCCTCTCCCTG-3`
		ep-Fc-Fw	5`-CCAGCCGGCCATGGCG-3`
ep-Fc-Rv	5`-GAATTCGGCCCCCGAGGCCCC-3`

Primers for cloning (SEQ ID NO: 1-27)

Example 3: searching for 2M libraries of Fc variants based on bacterial culture and flow cytometry

In this example, a 2M library of established Fc variants is retrieved. Specifically, 1mL of Fc variant library cells transformed into E.coli Jude1 cells were cultured with shaking in super broth (TB) medium supplemented with 2 wt/vol% glucose and chloramphenicol (40. Mu.g/mL) as an antibiotic at 37℃and 250rpm for 4 hours. After shaking culture, library cells were inoculated into TB medium at a ratio of 1:100 and shaking cultured at 250rpm and 37℃until OD ₆₀₀ reached 0.5. Thereafter, further incubation was carried out at 25℃for 20 minutes for cooling, and 1mM isopropyl-1-thio- β -D-galactoside (IPTG) was added to induce expression. After the completion of the incubation, the collected cells were divided into equal amounts based on OD ₆₀₀ normalization, and then centrifuged at 14000rpm for 1 minute. The harvested cells were resuspended in 1ml of 10mM Tris-HCl (pH 8.0) and washed twice by centrifugation for 1 min. Cells were resuspended in 1ml of STE (0.5M sucrose, 10mM Tris-HCl, 10mM EDTA (pH 8.0)) and centrifuged at 37℃for 30 minutes to remove the outer membrane. The supernatant was removed by centrifugation, 1ml of solution A (0.5M sucrose, 20mM MgCl ₂, 10mM MOPS (pH 6.8)) was added, followed by resuspension and centrifugation. Cells were resuspended in 1ml of a mixture of 1ml of solution A and 20. Mu.l of a50 mg/ml lysozyme solution, and then centrifuged at 37℃for 15 minutes to remove the peptidoglycan layer. The supernatant was removed and the cells were resuspended in 1ml of PBS. To 300. Mu.l of the suspension were added 700. Mu.l of PBS and fluorescent-labeled tetrameric FcgammaRIIIa-Alexa 488 fluorescent probe and centrifuged at room temperature to label the spheroplasts with the fluorescent probe. After labelling, the cells were washed once with 1ml of PBS and sorted by flow cytometry (S3 sortor (Bio-rad)) to collect the top 3% highly fluorescent cells. The sorted cells are reclassified for higher purity. For the re-sorted samples, the genes were amplified by PCR using Taq polymerase (Biosesang) and pMopac-seq-Fw and pMopac-seq-Rv primers, followed by a series of treatments including treatment with SfiI restriction enzyme, ligation and transformation to construct a sub-library in which the genes of the sorted cells were amplified. The procedure was performed for a total of 2 rounds. Thereafter, the 40 clones obtained were analyzed separately and M428L variants were selected which had a higher affinity for FcRn than the wild-type Fc at ph5.8 (fig. 3).

Example 3: construction of error and dot libraries of Fc variants

Two additional libraries were constructed using sorted M428L as template. First, mutations were introduced into the Fc by error-prone PCR to construct an error library. The library was constructed with an error rate of 0.3% error (2.04 bp) in Fc (680 bp) using ep-Fc-Fw and ep-Fc-Rv primers (size: 2X 10 ⁸). Next, M428L was used as a template for constructing the spot library. Libraries were constructed using pMopac12-seq-Fw、pMopac12-seq-Rv、Fc-Sub#0-Rv、Fc-Sub#1-1-Fw、Fc-Sub#1-2-Fw、Fc-Sub#1-3-Fw、Fc-Sub#1-4-Fw、Fc-Sub#1-5-Fw、Fc-Sub#1-Rv、Fc-Sub#2-1-Fw、Fc-Sub#2-2-Fw、Fc-Sub#2-3-Fw、Fc-Sub#2-Rv、Fc-Sub#3-1-Fw、Fc-Sub#3-2-Fw、Fc-Sub#3-3-Fw and Fc-sub#3-4-Fw primers to randomly introduce mutations into selected regions of Fc binding to FcRn (figure 4). Thereafter, a library of Fc variants was created by transformation to Jude1 in the same manner as described above.

Example 4: based on bacterial culture and flow cytometry, sorting of variants including PFc, PFc29, PFc, EFc29, EFc41, EFc82 and EFc88, error and spot libraries of Fc variants were retrieved

The sorting and reclassifying procedure described above was performed on other error libraries and spot libraries constructed based on sorted M428L. The error library was repeatedly sorted 5 rounds and reclassified, and the spot library was sorted only one round. A set of about 100 clones from each of the two libraries was analyzed separately and Fc variants were sorted with high affinity for FcRn at ph5.8 and low affinity for FcRn at ph 7.4. FACS analysis showed that EFc6, EFc29, EFc41, EFc46, EFc70, EFc90, EFc82 and EFc88 sorted from the error library showed higher fluorescence intensity at pH5.8 than wild-type Fc and include the conventional variants of YTE from Medimmune (Gabriel J. Robbie et al, antimicrob Agents chemther.2013, month 12; 57 (12): 6147-6153) and LS from Xencor (U.S. patent No. 8324351). EFc6, EFc29, EFc41, EFc82 and EFc88 were found to exhibit lower fluorescence intensities than LS at pH 7.4. Furthermore, PFc, PFc29, and PFc41 variants, which were selected from the spot library, showed higher fluorescence intensity than YTE and LS at pH 5.8. PFc30 shows a lower fluorescence intensity than YTE and LS at ph 5.8. PFc29 and PFc show lower fluorescence intensity than LS at pH 7.4. Finally, EFc6, EFc29, EFc41, EFc82, EFc88, PFc, PFc29, and PFc41 were chosen because they are expected to increase blood half-life (table 2 and fig. 5).

TABLE 2

Point mutations to sort variants

The positions of the mutations were numbered according to the Kabat EU numbering system, as described in Kabat et al, "Sequences of Proteins of Immunological Interest" 5 th edition, U.S. Pat. No. HEALTH AND Human Services, NIH Publication No.91-3242, 1991.

Example 5: generation and purification of control trastuzumab for introduction of Fc variants

Selection of representative IgG1 therapeutic antibody trastuzumabAs a control group. In the examples that follow, the sorted Fc variants were introduced into trastuzumab.

The heavy and light chain variable regions of wild-type trastuzumab were synthesized (Genscript) from the corresponding amino acid sequences from Drug Bank (http:// www.drugbank.ca /) by mammalian codon optimization and synchronous reverse translation. The synthetic trastuzumab heavy and light chain genes were subcloned into pOptiVEC-Fc and pdna3.3 vectors, respectively (fig. 6). Animal cell expression plasmids encoding trastuzumab heavy and light chains were prepared, expressed in HEK293 cells and purified.

After incubation in HEK 293F, wild-type trastuzumab was purified by protein a affinity chromatography (AKTA PRIME plus, cat No. 11001313) and gel permeation chromatography (HiTrap MabselectSure, GE, cat No. 11-0034-95). 7.7mg of wild-type trastuzumab was obtained in high purity from 300ml of medium (fig. 7).

Example 6: analysis and comparison of physical properties of internal trastuzumab and commercial trastuzumab

Unlike commercial trastuzumab produced by suspension culture in CHO cells, internal trastuzumab was produced in HEK 293. Two basic features of commercial trastuzumab and internal trastuzumab were analyzed before introduction and functional analysis of the sorted Fc variants. A pH gradient of 3 to 10 was established using Pharmalyte 3-10 carrier ampholyte (GE HEALTHCARE, 17-0456-01) and samples were analyzed for pI values and charge changes by capillary electrophoresis (CE: PA800Plus, beckman coulter). The analysis showed that no impurities were detected by size exclusion chromatography (SEC, tskgel G3000swxl, tosoh). For commercial trastuzumab, the pI value of the charge change was 8.27 to 8.74, with the pI of the main peak being 8.62. For internal trastuzumab, the pI value of the charge change was 8.29 to 8.78, the pI of the main peak was 8.65, almost identical to commercial trastuzumab (fig. 8). pI values were measured by capillary electrophoresis (CE, PA800Plus, beckman coulter). However, the cIEF analysis showed that the main and other peaks of internal trastuzumab were slightly different in content. These differences are due to the different glycan patterns of internal trastuzumab produced by the HEK293 cell line and commercial trastuzumab produced by the CHO cell line, which makes internal trastuzumab possible oxidation by sialic acid. Thus, glycan analysis was also performed (fig. 9).

After cleavage of N-glycans from proteins with PNG enzyme F (NEB, 186007990-1) and labelling with RapiFluor-MS reagent (Waters, 186007989-1), glycan analysis was performed using a UPLC system (acquisition UPLC class I, waters, FLR detector). The results of the glycan analysis showed that the glycan patterns were similar, but different constituent glycan contents were observed, which appeared to be caused not by sialic acid-induced oxidation but by different producer cell lines. Furthermore, glycans were found to have no significant effect on binding force analysis and pharmacokinetic analysis (data not shown). Thus, the sorted Fc variants were introduced into commercial trastuzumab and internal trastuzumab.

Example 7: preparation and purification of Fc variants and analysis of the physical Properties of Fc variants

Five control variants, including commercial wild-type variants, internal wild-type variants, LS (XenCor), YTE (MedImmune), and 428L, and sorted variants PFc, PFc41, EFc29, EFc41, and EFc82 were transfected into HEK293F animal cells. 300ml HEK293F cells were subcultured at a density of 1X 10 ⁶ cells/ml one day prior to transfection. The next day, cells were transfected with polyethylenimine (PEI, polyscience, 23966). First, the heavy chain gene and the light chain gene of each variant were mixed in a ratio of 2:1 in 30ml of Freestole 293 expression medium (Gibco, 12338-018). PEI and variant genes were then mixed in a 1:2 ratio, allowed to stand at room temperature for 20 minutes, mixed with cells subcultured the previous day, incubated in a CO ₂ shake incubator at 125rpm, 37℃and 8% CO ₂ for 6 days, and then centrifuged. Only the supernatant was collected.

Proteins were purified from the supernatant by affinity chromatography using AKTA prim plus and HiTrap MabselectSure columns. 300ml of the supernatant was passed through the column at a rate of 3 ml/min and washed with 100ml of 1 XPBS. Then, igG elution buffer (Thermo science, 21009) was flowed through the column at a rate of 5 ml/min. Six fractions (5 ml each) were collected. Each fraction was neutralized with 500. Mu.l of 1M Tris (pH 9.0). The protein in the fractions was assayed using Bradford (BioRad, 5000001) and placed in a new tube. The purified variants were concentrated using a 30K Amicon ultracentrifuge filter (UFC 903096) and analyzed for their physical properties (fig. 10 and 11).

Each Fc variant except for the internal wild-type trastuzumab was purified with protein A and its purity (. Gtoreq.90%) and molecular weight were determined by SDS-PAGE. High purity protein samples were obtained using SEC-HPLC (FIG. 11 a) for efficacy assessment and purity analysis (purity. Gtoreq.97%). Analysis under isocratic conditions (mobile phase 1 XPBS, pH7.0,1 ml/min flow rate) showed that all Fc variants had the same main peak retention time and molecular weights were estimated (FIG. 11 b).

Example 8: measurement of binding of Fc variants to FcRn by ELISA

ELISA was performed to measure pH-dependent binding of the prepared variants to FcRn and binding of the variants to fcγr and C1q, which binding renders the variants functional. First, the pH-dependent binding of variants to FcRn was studied. To this end, 50. Mu.l each of the IgG Fc variants was diluted to 4. Mu.g/ml with 0.05M Na ₂CO₃ (pH 9.6), fixed at 4℃on flat-bottomed polystyrene high-binding 96-well microplates (costar) for 16h, blocked with 100. Mu.l of 4% skim milk (GenomicBase) (in 0.05% PBST at pH5.8/pH 7.4) at room temperature for 2h, and then washed four times with 180. Mu.l of 0.05% PBST (pH 5.8/pH 7.4). After that, 50. Mu.l of FcRn was serially diluted with 1% skimmed milk (in 0.05% PBST at pH5.8/pH 7.4), added to each well and reacted at room temperature for 1h. After washing, the antibody was reacted with 50. Mu.l of anti-GST-HRP conjugate (GE HEALTHCARE) at room temperature for 1h. The plates were washed and developed with 50. Mu.l of 1-Step Ultra TMB-ELISA substrate solution (Thermo FISHER SCIENTIFIC). The reaction was stopped with 2M H ₂SO₄ (50. Mu.l each). The reaction products were then analyzed using an epoch microplate spectrophotometer (BioTek). The sorted variants had binding to FcRn similar to variant LS at ph5.8 and were more readily dissociated than LS at ph7.4 (fig. 12).

Example 9: measurement and comparison of trastuzumab Fc variants binding to monomeric hFcRn at ph6.0 and ph7.4

In this example, pH-dependent binding forces of commercial trastuzumab, internal trastuzumab, and sorted Fc variants that have been analyzed and studied for physical properties to human FcRn were compared. Specifically, K _D values were measured using a Biacore T200 instrument (GE HEALTHCARE). Human FcRn was used as an analyte in antigen-mediated antibody capture format at ph6.0 as disclosed in the literature (Yeung YA. et al, j.immunol, 2009). Each Fc variant as ligand was diluted in running buffer (50 mM phosphate, ph6.0, 150mM nacl,0.005% surfactant P20, ph 6.0), injected at a level of about 300 Response Units (RU) onto the surface of the CM5 chip, where HER2 ECD domain was immobilized to a level of about 3000RU and captured. To determine binding, monomeric FcRn as analyte (Sinobiological inc., CT 009-H08H) was serially diluted from 125nM FcRn running buffer, injected at a flow rate of 30 μl/min for 2min, and then dissociated for 2 min. In each cycle, 10mM glycine (pH 1.5) was used for regeneration at a flow rate of 30 ml/min for 30 seconds. The sensorgrams were fitted to a 1:1 binding model using BIAevaluation software (Biacore). As a result, the Fc variants had higher binding capacity (PFc 3:5.6nM, PFc29:6.8nM, PFc41:5.9nM, etc.) compared to commercial trastuzumab (15 nM) and internal trastuzumab (16.9 nM) as control groups and 428L (9 nM) as main chain. But Fc variants bind less than YTE (5.7 nM) and LS (4.1 nM), which are the highest values in the known world, but differ almost equally within the error range. Dissociation was assessed using the avid format, in which monomeric hFcRn was directly immobilized and different concentrations of Fc variants were infused (Zalevsky J et al nat. Biotechnol, 2010), since the ligand bound less to the analyte at pH 7.0. The human FcRn ECD domain (Sino Biological) was immobilized to the CM5 chip surface at a level of about 1500 RU. The Fc variants were serially diluted in HBS-EP (pH 7.4) by 3000nM and injected onto the FcRn-immobilized chip surface at a flow rate of 5 ml/min for 2 min. Bound Fc variants were dissociated for 2 minutes. After each cycle was completed, the chip surface was regenerated (contact time 30 seconds; flow rate 30. Mu.l/min) with 100mM Tris (pH 9.0). In particular, fc variants PFc and PFc41 retain their high binding capacity at ph6.0 and dissociate more rapidly at ph7.4 than YTE and LS. These results are consistent with those obtained by ELISA and indicate the expected long half-life of the Fc variants (fig. 13). In practice, in vivo pharmacokinetic experiments were performed in human FcRn Tg mice.

Example 10: analysis and comparison of in vivo PK experiments with commercial trastuzumab and internal trastuzumab in conventional B6 mice and hFcRn Tg mice

PK analysis was performed in a conventional B6 mouse (Jungang laboratory animal resources center, C57BL/6J (B6)) with the same genetic background as the human FcRn Tg mouse. As a result, as reported in the literature, the Fc of human antibodies was found to have a higher affinity for conventional mouse FcRn than for human FcRn. In conventional mice, there was a difference in PK values between the internal and commercial antibodies, and the internal antibodies appeared to be unstable. In addition, the AUC of Tg mice (b 6.Cg-Fcgrttm1DCR PRKDCSCID TG (Jackson laboratories, CAGFCGRT) 276 Dcr/DcrJ) was slightly lower than that of conventional mice, but the pharmacokinetic profiles of the internal and commercial antibodies were similar. This suggests that no problem was encountered in experiments using internal Fc variants generated in HEK293 to analyze the actual in vivo pharmacokinetics of Fc variants in Tg mice (fig. 14).

Example 11: four types of pharmacokinetics in hFcRn Tg mice including LS, YTE and variants PFc, 29 and PFc41

The binding force measured using ELISA system and BiaCore instrument at pH6.0 and pH7.4 was found to be constant. From these results, since the binding forces of PFc and PFc were comparable to those of LS under acidic conditions (pH 6.0) and the dissociation forces of PFc and PFc41 were higher than those of LS at pH7.4, sorting was performed on PFc and PFc 41. Meanwhile, LS mutant of Xencor and YTE of MedImmune, which are currently known to be the most potent in the world, were used as control groups, and two trastuzumab Fc variants were injected into 20 human FcRn Tg mice (5 animals per group, 5mg/kg I.V (tail vein)). Following injection, blood samples were taken 12 times (0 min, 30 min, 1 hr, 6 hr, 24 hr, 3 days, 7 days, 14 days, 21 days, 28 days, 35 days, 42 days and 50 days) from the facial vein. Blood samples were analyzed for Fc variant concentration by ELISA, followed by non-compartmental analysis (NCA) using WinNonlin. Based on the results of ELISA and BiaCore assays, fc variants PFc and PFc41 exhibited increased in vivo half-life as expected. In particular, PFc has a longer half-life than conventional LS (FIG. 15 and Table 3).

TABLE 3

Non-atrioventricular analysis of pharmacokinetic parameters following intravenous administration of trastuzumab Fc variants to mice (5 mg/kg) (data expressed as mean ± SD (n=5)).

T _1/2, terminal half-life; t _{Maximum value} , time of maximum concentration; c ₀, estimating the concentration at zero time; c _{Maximum value} , maximum concentration; AUC _{Final result} , the area under the curve from the point of administration to the final measured concentration; AUC _{Infinity is provided} , the area under the curve from the point of administration to infinity; AUC _{％ Inference} , the inferred area under the curve as a percentage of the area under the total curve; v _z, dispense volume; CL, clear.

Example 12: measurement of binding force of Fc variants to FcgammaR by ELISA

In this example, binding of Fc variants to fcγr was measured. Specifically, 50. Mu.l each of the IgG Fc variants was diluted to 4. Mu.g/ml with 0.05M Na ₂CO₃ (pH 9.6), fixed on flat-bottomed polystyrene high-binding 96-well microplates (costar) at 4℃for 16h, blocked with 100. Mu.l of 4% skimmed milk (GenomicBase) (in 0.05% PBST at pH 7.4) at room temperature for 2h, and washed four times with 180. Mu.l of 0.05% PBST (pH 7.4). Thereafter, 50. Mu.l of FcgammaR was serially diluted with 1% skim milk (in 0.05% PBST at pH 7.4), added to each well, and reacted at room temperature for 1h. After washing, the antibody was reacted with 50. Mu.l of anti-GST-HRP conjugate (GE HEALTHCARE) at room temperature for 1h. The plates were washed and developed with 50. Mu.l of 1-Step Ultra TMB-ELISA substrate solution (Thermo FISHER SCIENTIFIC). The reaction was stopped with 2M H ₂SO₄ (50. Mu.l each). The reaction products were then analyzed using an epoch microplate spectrophotometer (BioTek). Each experiment was repeated twice. Fig. 16 shows binding force of Fc variants to fcγr (fcγri, fcγriia (H), fcγriia (R), fcγriib, fcγriiia (V), and fcγriiia (F)) measured by ELISA.

Example 13: measurement of effector function of antibody-dependent cell-mediated cytotoxicity (ADCC) of Fc variants

Antibody Dependent Cellular Cytotoxicity (ADCC) activity of trastuzumab Fc variants was assessed using an ADCC reporter bioassay kit (Promega, G7010). Specifically, SKBR-3 cells as target cells were seeded at a density of 5X 10 ³ cells/100. Mu.l in each well of a 96-well tissue culture plate and cultured in a CO ₂ incubator at 37℃for 20 hours. Thereafter, 95 μl of culture medium was removed from each well of the plate using a multi-cartridge pipette, and 25 μl of ADCC assay buffer provided by the ADCC reporter bioassay kit was added to each well. Normal IgG, trastuzumab and trastuzumab Fc variants were diluted to various concentrations with ADCC assay buffer. Mu.l of each dilution was added to each well of a 96-well tissue culture plate containing cells and allowed to stand at room temperature until effector cells were added. Effector cells provided by the kit were lysed in a constant temperature water bath at 37 ℃ for 2 to 3 minutes, and then 630 μl of the solution was mixed with 3.6mL of ADCC assay buffer. Mu.l of effector cells were added to each well of the plate containing target cells and antibody dilutions. The reaction was carried out in a CO ₂ incubator at 37℃for 6h. After a predetermined time, the plate was taken out of the incubator and left to stand at room temperature for 15 minutes. Mu.l of Bio-Glo ^TM luciferase assay reagent was added to each well and reacted at room temperature for 5 minutes. After the reaction was completed, luminescence of each well was measured using a photometer (Enspire multi-mode plate reader). ADCC activity of each test antibody was determined by expressing the average of the experimental results as fold induction calculated from the following formula:

Fold induction = RLU (induction ¹ -background ²)/RLU (no antibody control ³ -background)

Induction ¹: RLU values obtained from samples comprising target cells, test antibodies and effector cells

Background ²: RLU values obtained from ADCC assay buffer

Antibody-free control ³: RLU values obtained from samples containing only target cells and effector cells

The ADCC activity of trastuzumab Fc variants (LS, YTE, PFC and PFC 41) against SKBR-3 was compared to the ADCC activity of trastuzumab against SKBR-3 (FIG. 17). As a result, the maximum ADCC activity of LS, PFc29 and PFc41 at the highest concentration was about 1.5 times, about 1.18 times and about 1.27 times, respectively, that of trastuzumab as a positive control group. In contrast, the maximum ADCC activity of YTE at the highest concentration was about 4.2 times that of trastuzumab. In summary, the ADCC activity of trastuzumab Fc variants PFc and PFc41, as well as control variant LS, was 1.18-1.5-fold that of control trastuzumab. EC50 values of the variants were measured. As shown in fig. 18, the EC50 values of Fc variants PFc29 (0.04543 μg/mL) and PFc (0.05405 μg/mL) were lower compared to control LS (0.05575 μg/mL), indicating that the efficacy of Fc variants PFc29 and PFc41 were more stable.

Example 14: measurement of binding force of Fc variants to C1q by ELISA

In this example, the binding of the Fc variant to C1q was measured. Specifically, 50. Mu.l each of the IgG Fc variants was diluted to 4. Mu.g/ml with 0.05MNA ₂CO₃ (pH 9.6), fixed at 4℃on flat-bottomed polystyrene high-binding 96-well microplates (costar) for 16h, blocked with 100. Mu.l of 4% skim milk (GenomicBase) (in 0.05% PBST at pH 7.4) at room temperature for 2h, and washed four times with 180. Mu.l of 0.05% PBST (pH 7.4). Thereafter, 50. Mu.l of complementary human C1q (Millipore) was serially diluted with 1% skim milk (in 0.05% PBST at pH 7.4), added to each well, and reacted at room temperature for 1h. After washing, the reaction of the antibody with 50. Mu.l of anti-C1 q-HRP conjugate (Invitrogen) was carried out at room temperature for 1h. The plates were washed and developed with 50. Mu.l of 1-Step Ultra TMB-ELISA substrate solution (Thermo FISHER SCIENTIFIC). The reaction was stopped with 2M H ₂SO₄ (50. Mu.l each). The reaction products were then analyzed using an epoch microplate spectrophotometer (BioTek). The results of the analysis showed that the binding force of sorted PFc to C1q was higher than that of conventional LS and YTE to C1q (FIG. 19).

While specific embodiments of the invention have been described in detail, it will be apparent to those skilled in the art that these specific embodiments are merely preferred embodiments and are not intended to limit the scope of the invention. Accordingly, the true scope of the invention should be defined by the following claims and their equivalents.

Claims (12)

1. A polypeptide comprising a variant Fc of a human IgG1 antibody, wherein said variant Fc has an increased half-life compared to the wild type,

Wherein the half-life is increased by amino acid substitutions in the Fc domain of the wild-type human antibody,

Wherein the half-life increasing amino acid substitutions are M428L and Q311R or M428L and L309G according to the Kabat EU numbering system.

2. An antibody comprising the polypeptide of claim 1.

3. The antibody of claim 2, wherein the antibody is a polyclonal antibody, a monoclonal antibody, a minibody, a domain antibody, a bispecific antibody, an antibody mimetic, a chimeric antibody, an antibody conjugate, a human antibody, a humanized antibody, or a fragment thereof.

4. A nucleic acid molecule encoding the polypeptide of claim 1.

5. A vector comprising the nucleic acid molecule of claim 4.

6. A host cell comprising the vector of claim 5.

7. A composition comprising the polypeptide of claim 1, the antibody of claim 2, the nucleic acid molecule of claim 4, or the vector of claim 5.

8. The composition of claim 7, wherein the composition increases the blood half-life of a therapeutic antibody or protein.

9. The composition of claim 7, wherein the composition is a pharmaceutical composition for preventing or treating cancer.

10. The composition of claim 9, wherein the composition recognizes a cancer antigen.

11. A method for producing a polypeptide comprising a human antibody Fc variant having an increased half-life compared to wild-type, the method comprising: a) Culturing a host cell comprising a vector comprising a nucleic acid molecule encoding the polypeptide of claim 1; and b) harvesting the polypeptide expressed by the host cell.

12. A method for producing an antibody having an increased half-life compared to wild-type, comprising: a) Culturing a host cell that expresses an antibody comprising the polypeptide of claim 1; and b) purifying the antibody expressed by the host cell.